There is currently intensive research on the design of novel human immunodeficiency virus type 1 (HIV-1) vaccine immunogens that can elicit potent neutralizing antibodies. A prerequisite for comparing and optimizing these strategies is the ability to precisely measure neutralizing antibody responses. To this end, we sought to develop new single-round HIV-1 infection assays to accurately measure HIV-1 neutralization. The precision and reproducibility of these assay will facilitate the measurement of HIV-1 neutralization, particularly incrementally improved neutralizing antibody responses generated by new candidate vaccines.